IN VITRO CYCLOSPORINE TOXICITY

Abstract

The epithelial cell line LLC-PK1, which expresses many proximal tubular characteristics, was used to investigate the relationship between calcium, the calcium channel blocker verapamil, and cyclosporine toxicity. The LLC-PK1 cells took up cyclosporine when this was added in a concentration of 2 micrograms/ml, and this uptake was maximal at 30 min (112 +/- 3 ng cyclosporine/mg cell protein). At 12 micrograms/ml it inhibited the sodium glucose cotransporter, as assessed by phlorizin-inhibitable 14C-alpha-methyl glucopyranoside (alpha-MG) uptake (control 37.2 +/- 6.3, 12 micrograms/ml 21.2 +/- 1.1 mumol/hr/mg protein). Cyclosporine at 2 micrograms/ml did not affect cell growth after 5 days (control 945 +/- 60 micrograms cell protein per 25 cm2 flask, 2 micrograms/ml cyclosporine/ml 1046 +/- 32 micrograms protein/flask), even in the presence of 7.6 mM ionized calcium (862 +/- 37 micrograms protein/flask). Cyclosporine at 12 micrograms/ml inhibited cell growth (286 +/- 27 micrograms protein/flask), and raising the ambient ionized calcium concentration to 7.6 mM reduced cell growth further (91 +/- 6 micrograms protein/flask). Cyclosporine at concentrations of 2 and 12 micrograms/ml produced increasing cell vacuolation, as seen in vivo. Short-term uptake of 2 micrograms/ml cyclosporine could be inhibited by 1.0 mM and 0.5 mM verapamil (49 +/- 9.5 and 71 +/- 6.4 ng cyclosporine/mg cell protein, respectively, at 30 min). However, in the presence of 2 micrograms/ml cyclosporine 0.1 mM verapamil was toxic to the cells grown over five days (44 +/- 5 micrograms protein/flask). At 0.01 mM verapamil was not toxic to cell growth (921 +/- 29 micrograms protein/flask), but raising the medium calcium to 7.6 mM reduced cell growth (652 +/- 96 micrograms/ml). Inhibition of cyclosporine uptake did not occur with 0.01 mm verapamil (control 145.6 +/- 12.3 vs. 0.01 mM verapamil 150.4 +/- 3.8 ng cyclosporine/mg cell protein). The LLC-PK1 cell line represents a good in vitro model for cyclosporine renal tubular toxicity, as the in vivo observation of glycosuria and proximal tubular cell vacuolation in cyclosporine nephrotoxicity can be reproduced. In vitro this is shown to be associated with inhibition of sodium-dependent glucose cotransport. Verapamil inhibited cyclosporine uptake, but only at concentrations that were toxic to the cells. Verapamil potentiated rather than reduced the increased cyclosporine toxicity produced by increasing the medium calcium concentration. The suggested protective effect of verapamil against cyclosporine nephrotoxicity is therefore unlikely to be due to inhibition of cyclosporine uptake or of calcium entry into proximal tubular cells.